Skip to main content
. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Cancer Res. 2010 May 11;70(11):4375–4384. doi: 10.1158/0008-5472.CAN-09-4403

Figure 2. ITGA7 induces MCM7 phosphorylation is ILK dependent.

Figure 2

(A) ITGA7 induces basic myelin phosphorylation by ILK immunoprecipitates. PITT1 or PITT2 cells were induced with or without 5 μg/ml tetracycline. ILK was immunoprecipitated. Basic myelin phosphorylation by ILK was performed through ELISA assay. (B) Induction of MCM7 phosphorylation by ITGA7 in PITT1 and PITT2 cells. PITT1 or PITT2 cells were induced with or without 5 μg/ml tetracycline. MCM7 was immunoprecipitated, electrophoresed in 8% SDS-PAGE and immunoblotted with antibodies against phospho-serine, phospho-threonine and MCM7. The top two panels are protein extracts immunoblotted with anti-ITGA7 and β-actin antibodies. (C) ILK is required for MCM7 phosphorylation in vivo and in vitro. Left panel: Knocking down of ILK abrogates MCM7 phosphorylation induced by ITGA7. PITT1 cells were transfected with siRNA specific for ILK (lanes 1–2) or scramble control (lanes 3–4), and induced with or without tetracycline. The MCM7 immunoprecipitates were immunbloted with the indicated antibodies. The top two panels are protein extracts immunoblotted with anti-ITGA7 and β-actin antibodies. Right panel: Decrease of MCM7 phosphorylation in H358 and H1299 cells with siRNA specific for ILK. Top two panels: Protein extracts from H358 and H1299 cells treated with scramble siRNA (Scr) or ILK siRNA (sILK) were SDS-PAGED and immunoblotted with antibodies specific for ILK and β-actin. Bottom three panels: Immunoprecipitates of MCM7 from H358 and H1299 cells were immunoblotted with antibodies specific for MCM7, phospho-serine, and phospho-threonine. (D) GST-MCM7 N-terminus phosphorylated by ILK immunoprecipitates. Kinase assays were performed on ILK immunoprecipitates using GST or GST-MCM7n as substrate. GST and GST-MCM7n were electrophoresed in 10% SDS-PAGE and immunoblotted with antibodies specific for phospho-serine or phospho-threonine. Ab- denotes protein extract pre-cleared by ILK antibody column before immunoprecipitation.